Isolevuglandin-protein adducts in humans: products of free radical-induced lipid oxidation through the isoprostane pathway

A family of extremely reactive electrophiles, isolevuglandins (isoLGs), is generated in vivo by free radical-induced lipid oxidation and rearrangement of endoperoxide intermediates of the isoprostane pathway. Protein adducts of two different oxidized lipids, isoLGE(2) and iso[4]LGE(2), and the corresponding autoantibodies are present in human blood. Western blot analysis of a polyacrylamide gel electrophoresis gel detects several immunoreactive plasma proteins. Only a minor fraction of the isoLG-protein modifications is associated with low density lipoprotein since mean levels were decreased only 20-22% by immunoprecipitation of apolipoprotein B (apoB). Mean levels of both isoLGE(2) and iso[4]LGE(2)-protein adducts in plasma from patients with atherosclerosis (AS) (n=16) or end-stage renal disease (RD) (n=8) are about twice those in healthy individuals (n=25). These elevated levels are not related to variations in age, total cholesterol or apoB. A linear correlation (r=0.79) between plasma isoLGE(2) and iso[4]LGE(2)-protein adduct levels in all 49 individuals is consistent with a common free radical-induced mechanism for the production of both oxidized lipids in vivo. The correlation is even stronger (r=0.86) for patients with AS or RD. That isoLG-protein adduct levels are more strongly correlated with disease than are total cholesterol or apoB suggests an independent defect that results in an abnormally high level of oxidative injury associated with AS and RD.     

Protein adducts of iso[4]levuglandin E2, a product of the isoprostane pathway, in oxidized low density lipoprotein.

Levuglandin (LG) E2, a cytotoxic seco prostanoic acid co-generated with prostaglandins by nonenzymatic rearrangements of the cyclooxygenase-derived endoperoxide, prostaglandin H2, avidly binds to proteins. That LGE2-protein adducts can also be generated nonenzymatically is demonstrated by their production during free radical-induced oxidation of low density lipoprotein (LDL). Like oxidized LDL, LGE2-LDL, but not native LDL, undergoes receptor-mediated uptake and impaired processing by macrophage cells. Since radical-induced lipid oxidation produces isomers of prostaglandins, isoprostanes (isoPs), via endoperoxide intermediates, we postulated previously that a similar family of LG isomers, isoLGs, is cogenerated with isoPs. Now iso[4]LGE2-protein epitopes produced by radical-induced oxidation of arachidonic acid in the presence of protein were detected with an enzyme-linked immunosorbent assay. Iso[4]LGE2-protein epitopes are also generated during free radical-induced oxidation of LDL. All of the LGE2 isomers generated upon oxidation of LDL are efficiently sequestered by covalent adduction with LDL-based amino groups. The potent electrophilic reactivity of iso-LGs can be anticipated to have biological consequences beyond their obvious potential as markers for specific arachidonate-derived protein modifications that may be of value for the quantitative assessment of oxidative injury.     

Isolevuglandin-modified proteins, including elevated levels of inactive calpain-1, accumulate in glaucomatous trabecular meshwork

We report that protein adducts of iso[4]levuglandin E2 (iso[4]LGE2), a highly reactive product of free radical-induced lipid oxidation, accumulate in human glaucomatous trabecular meshwork (TM) but not in controls. Reactive oxygen species play a pathogenic role in primary open angle glaucoma by fostering changes that reduce permeability of the TM tissue and consequently impede aqueous humor outflow resulting in elevated intraocular pressure. IsoLGs covalently modify proteins and are especially effective in causing protein-protein cross-linking. We found elevated levels of calpain-1 in glaucomatous TM. However, calpain activity in glaucomatous TM is only about 50% of that in controls. This paradox is explicable by the fact that modification by isoLGs renders calpain-1 inactive. Thus, treatment of calpain-1 with iso[4]LGE2 in vitro results in covalent modification, inactivation, the formation of high molecular weight aggregates (as determined by Western and dynamic light scattering analyses), and resistance to proteasomal digestion. Iso[4]LGE2-modified calpain-1 undergoes ubiquitination, and its loading impairs the cellular proteasome activity, consistent with competitive inhibition and formation of suicidal high molecular weight aggregates. These data suggest that interference with proteasomal activity, owing to protein modification by isoLGs, could contribute to glaucoma pathophysiology by decreasing the ability of the TM to modulate outflow resistance.     

Formation of highly reactive gamma-ketoaldehydes (neuroketals) as products of the neuroprostane pathway.

Neuroprostanes are prostaglandin-like compounds produced by free radical-induced peroxidation of docosahexaenoic acid, which is highly enriched in the brain. We previously described the formation of highly reactive gamma-ketoaldehydes (isoketals) as products of the isoprostane pathway of free radical-induced peroxidation of arachidonic acid. We therefore explored whether isoketal-like compounds (neuroketals) are also formed via the neuroprostane pathway. Utilizing mass spectrometric analyses, neuroketals were found to be formed in abundance in vitro during oxidation of docosahexaenoic acid and were formed in greater abundance than isoketals during co-oxidation of docosahexaenoic and arachidonic acid. Neuroketals were shown to rapidly adduct to lysine, forming lactam and Schiff base adducts. Neuroketal lysyl-lactam protein adducts were detected in nonoxidized rat brain synaptosomes at a level of 0.09 ng/mg of protein, which increased 19-fold following oxidation in vitro. Neuroketal lysyl-lactam protein adducts were also detected in vivo in normal human brain at a level of 9.9 +/- 3.7 ng/g of brain tissue. These studies identify a new class of highly reactive molecules that may participate in the formation of protein adducts and protein-protein cross-links in neurodegenerative diseases and contribute to the injurious effects of other oxidative pathologies in the brain.     

Isolevuglandins and mitochondrial enzymes in the retina: mass spectrometry detection of post-translational modification of sterol-metabolizing CYP27A1

We report the first peptide mapping and sequencing of an in vivo isolevuglandin-modified protein. Mitochondrial cytochrome P450 27A1 (CYP27A1) is a ubiquitous multifunctional sterol C27-hydroxylase that eliminates cholesterol and likely 7-ketocholesterol from the retina and many other tissues. We investigated the post-translational modification of this protein with isolevuglandins, arachidonate oxidation products. Treatment of purified recombinant CYP27A1 with authentic iso[4]levuglandin E(2) (iso[4]LGE(2)) in vitro diminished enzyme activity in a time- and phospholipid-dependent manner. A multiple reaction monitoring protocol was then developed to identify the sites and extent of iso[4]LGE(2) adduction. CYP27A1 exhibited only three Lys residues, Lys(134), Lys(358), and Lys(476), that readily interact with iso[4]LGE(2) in vitro. Such selective modification enabled the generation of an internal standard, (15)N-labeled CYP27A1 modified with iso[4]LGE(2), for the subsequent analysis of a human retinal sample. Two multiple reaction monitoring transitions arising from the peptide AVLK(358)(-C(20)H(26)O(3))ETLR in the retinal sample were observed that co-eluted with the corresponding two (15)N transitions from the supplemented standard. These data demonstrate that modified CYP27A1 is present in the retina. We suggest that such protein modification impairs sterol elimination and likely has other pathological sequelae. We also propose that the post-translational modifications identified in CYP27A1 exemplify a general mechanism whereby oxidative stress and inflammation deleteriously affect protein function, contributing, for example, to cholesterol-rich lesions associated with age-related macular degeneration and cardiovascular disease. The proteomic protocols developed in this study are generally applicable to characterization of lipid-derived oxidative protein modifications occurring in vivo, including proteins bound to membranes.     

Identification of extremely reactive gamma-ketoaldehydes (isolevuglandins) as products of the isoprostane pathway and characterization of their lysyl protein adducts

Isoprostanes are prostaglandin-like compounds produced by non-enzymatic peroxidation of arachidonic acid. The cyclooxygenase-derived endoperoxide, prostaglandin H2, can undergo rearrangement to highly reactive gamma-ketoaldehyde secoprostanoids (levuglandin E2 and D2). We explored whether isoprostane endoperoxide intermediates also rearrange to levuglandin-like compounds (isolevuglandins). Formation of a series of isolevuglandins during oxidation of arachidonic acid in vitro was established utilizing a number of mass spectrometric analyses. However, these compounds could not be detected in free form in protein-containing biological systems, which we hypothesized was due to extremely rapid adduction to amines. This was supported by the finding that >60% of levuglandin E2 adducted to albumin within 20 s, whereas approximately 50% of 4-hydroxynonenal still remained unadducted after 1 h. By utilizing electrospray tandem mass spectrometry, we established that these compounds form oxidized pyrrole adducts (lactams and hydroxylactams) with lysine. Formation of isolevuglandin-lysine adducts on apolipoprotein B was readily detected during oxidation of low density lipoprotein following enzymatic digestion of the protein to single amino acids. These studies identify a novel series of extremely reactive products of the isoprostane pathway that rapidly form covalent adducts with lysine residues on proteins. This provides the basis to explore the formation of isolevuglandins in vivo to investigate the potential biological ramifications of their formation in settings of oxidant injury.     

Modification of proteins by isoketal-containing oxidized phospholipids

Oxidative stress frequently leads to altered function of membrane proteins. Isoketals are highly reactive products of the isoprostane pathway of free radical-induced lipid peroxidation that rapidly form covalent protein adducts and exhibit a remarkable proclivity to form protein cross links in vitro. Examination of isoketal adducts from an animal model of oxidative injury revealed that initial adducts were formed by isoketals esterified in phospholipids, representing a novel oxidative injury-associated modification of proteins by phospholipids. Maturation of adducts involved cleavage from phospholipids and conversion of adducts to a more stable chemical form that can be detected for extended periods. Because initial adducts were formed by phospholipid-esterified isoketals, the functional consequence of isoketal adduction was examined using a model membrane protein (a cardiac K(+) channel). These studies revealed that isoketal adduction profoundly altered protein function, inhibiting potassium current in a dose-dependent manner. These findings indicate that phospholipid-esterified isoketals rapidly adduct membrane proteins and that such modification can alter protein function, suggesting a generalized cellular mechanism for alteration of membrane function as a consequence of oxidative stress.     

Semotiadil, a new calcium antagonist, is a very potent inhibitor of LDL-oxidation

The influence of semotiadil, a novel benzothiazine calcium antagonist on in-vitro copper-, 2,2àzo-bis-(2,4 dimethylvaleronitrile)[AMVN]-, and 2,2àzo-bis-(2-amidinopropane) [AAPH]-induced low-density lipoprotein (LDL) oxidation was assessed. The following parameters were measured: lag-time of oxidation, maximal rate of oxidation, dienes formed through continuous monitoring of developing conjugated dienes, thiobarbituric acid reactive substances, isoprostane(8-iso-PGF2alpha)-generation and relative electrophoretic mobility. The effect was compared with nifedipine, amlodipine and diltiazem. Besides the influence on isoprostane (8-iso-PGF2alpha)-generation where nifedipine was equipotent with semotiadil at 10(-3) M, semotiadil demonstrated a strong and significant effect in attenuating the indicated indices of LDL-oxidation, in particular, dose-dependently (10(-3) M to 10(-7) M). These results indicate that semotiadil may have the strongest antioxidant activity on LDL among the calcium antagonists examined.     

F(2)-isoprostane and prostaglandin F(2 alpha)metabolite excretion rate and day to day variation in healthy humans.

Isoprostanes are mainly formed in vivo by a non-enzymatic free radical catalysed oxidation of arachidonic acid. Studies have indicated that a major isoprostane, 8-iso-PGF(2 alpha)in plasma and urine is a reliable biomarker of oxidative stress. Prostaglandins are formed by enzymatic oxidation of arachidonic acid catalysed by cyclooxygenase (COX). 15-Keto-dihydro-PGF(2 alpha), a major metabolite of prostaglandin F(2 alpha)in plasma, and also found in urine, is considered to be a useful biomarker of inflammation. To investigate the excretion pattern and day to day variation of 8-iso-PGF(2 alpha)and 15-keto-dihydro-PGF(2 alpha)in healthy individuals, morning urine samples were collected from 13 volunteers on 10 successive days. The samples were analysed for free 8-iso-PGF(2 alpha)and 15-keto-dihydro-PGF(2 alpha)by radioimmunoassay. The mean excretion rate of 8-iso-PGF(2 alpha)was 0.27+/-0.11 nmol/mmol creatinine (mean+/-SD, n=13) and the coefficient of variation was 42% during the 10 days. The mean excretion rate of 15-keto-dihydro-PGF(2 alpha)was 0.46+/-0.19 nmol/mmol creatinine, giving a coefficient of variation of 41%. The mean values of 8-iso-PGF(2 alpha)were significantly correlated with the mean values of 15-keto-dihydro-PGF(2 alpha)(r=0.68, P=0.01). In conclusion, day to day biological variation in urinary excretion rate of 8-iso-PGF(2 alpha)and 15-keto-dihydro-PGF(2 alpha)should be taken into account in evaluating a clinical study unless a large increase or decrease of these parameters has been obtained.     

Accumulation of 15-deoxy-delta(12,14)-prostaglandin J2 adduct formation with Keap1 over time: effects on potency for intracellular antioxidant defence induction

The COX (cyclo-oxygenase) pathway generates the reactive lipid electrophile 15d-PGJ2 (15-deoxy-Delta(12,14)-prostaglandin J2), which forms covalent protein adducts that modulate cell signalling pathways. It has been shown that this regulates important biological responses, including protection against oxidative stress, and supports the proposal that 15d-PGJ2 has pharmacological potential. Protective pathways activated by 15d-PGJ2 include those controlling the synthesis of the intracellular antioxidants GSH and the enzyme HO-1 (haem oxygenase-1). The induction of the synthesis of these intracellular antioxidants is, in large part, regulated by covalent modification of Keap1 (Kelchlike erythroid cell-derived protein with 'capn'collar homologyassociated protein 1) by the lipid and the subsequent activation of the EpRE (electrophile-response element). For the first time, we show that the potency of 15d-PGJ2 as a signalling molecule in endothelial cells is significantly enhanced by the accumulation of the covalent adduct with 15d-PGJ2 and endogenous Keap1 over the time of exposure to the prostaglandin. The consequence of this finding is that signalling initiated by electrophilic lipids differs from agonists that do not form covalent adducts with proteins because the constant generation of very lowconcentrations of 15d-PGJ2 can lead to induction of GSH or HO-1. In the course of these studies we also found that a substantial amount (97-99%) of exogenously added 15d-PGJ2 is inactivated in the medium and does not enter the cells to initiate cell signalling. In summary, we propose that the accumulation of covalent adduct formation with signalling proteins provides a mechanism through which endogenous intracellular formation of electrophilic lipids from COX can exert an anti-inflammatory effect in vivo.     

New developments in the isoprostane pathway: identification of novel highly reactive gamma-ketoaldehydes (isolevuglandins) and characterization of their protein adducts.

The bicyclic endoperoxide prostaglandin (PG) H2 undergoes nonenzymatic rearrangement not only to PGE2 and PGD2, but also to levuglandins (LG) E2 and D2, which are highly reactive gamma-ketoaldehydes. Isoprostanes (IsoPs) are PG-like compounds that are produced by nonenzymatic peroxidation of arachidonic acid. PGH2-like endoperoxides are intermediates in this pathway. Therefore, we explored whether the IsoP endoperoxides also undergo rearrangement to form IsoLGs. Oxidation of arachidonic acid in vitro resulted in the formation of abundant quantities of compounds that were established to be IsoLGs by using mass spectrometric analyses. However, the formation of IsoLGs could not be detected in biological systems subjected to an oxidant stress. We hypothesized that this was due to extremely rapid adduction of IsoLGs to proteins. This notion was supported by the finding that LGE2 adducted to albumin at a rate that exceeded that of 4-hydroxynonenal by several orders of magnitude: >50% of LGE2 had adducted within 20 s. We therefore undertook to characterize the nature of LG adducts. Using liquid chromatography electrospray tandem mass spectrometry, we established that LGs form oxidized pyrrole adducts (lactams and hydroxylactams) with the epsilon-amino group of lysine. Oxidation of low density lipoprotein resulted in readily detectable IsoLG adducts on apolipoprotein B after enzymatic digestion of the protein to individual amino acids. These studies identify a novel class of ketoaldehydes produced by the IsoP pathway that form covalent protein adducts at a rate that greatly exceeds that of other known aldehyde products of lipid peroxidation. Elucidation of the nature of the adducts formed by IsoLGs provides the basis to explore the formation of IsoLGs in vivo and investigate the potential biological ramifications of their formation in settings of oxidant injury.     

Selective stimulation of in situ intermediary metabolism by free calcium in permeabilized rat adipocytes.

The hypothesis that ionized calcium [Ca2+]i may stimulate in situ rat adipocyte intermediary metabolism distal to glucose transport was tested. A metabolically active porous adipocyte model was employed in which pathway metabolism is exclusively pore-dependent using glucose 6-phosphate (G6P) as substrate. Cellular [Ca2+]i was, furthermore, directly adjusted to between 0-2.5 microM via the membrane pores. Three metabolic fluxes were examined, (1) glycolysis-Krebs ([6-14C]G6P oxidation), (2) glycolysis to lactate ([U-14C]G6P to [14C]lactate) and (3) pentose pathway ([1-14C]G6P oxidation). Glycolysis-Krebs oxidation was was found to be selectively (33% above basal P less than 0.001) stimulated by 0.625 microM free calcium. In contrast, there was no effect of [Ca2+]i on the other, exclusively cytoplasmic, pathways. The stimulation of glycolysis-Krebs by [Ca2+]i was inhibited by a mitochondrial calcium channel blocker (Ruthenium red) and persisted over a range of ATP/ADP ratios. Separate studies demonstrated that 2-[1-14C]ketoglutarate oxidation was also calcium-stimulated in the porous adipocytes (160% over baseline at 1 microM [Ca2+]i). These studies thus demonstrate that physiologically relevant increments in porous adipocyte [Ca2+]i enhance overall in situ glycolytic-Krebs pathway oxidation by a mechanism which entails mitochondrial calcium uptake. Methodologically, this metabolically active porous adipocyte model presents a novel experimental approach to investigations regarding the effects of ionized calcium on intermediary metabolism beyond glucose transport.     

Both cyclooxygenase- and cytokine-mediated inflammation are associated with carotid intima-media thickness

BACKGROUND: Common carotid artery intima-media thickness (CCA-IMT) is a valid index of atherosclerosis, which is viewed as an inflammatory disease. It is unknown if various modes of inflammation (cyclooxygenase [COX]-mediated, cytokine-mediated), oxidative stress and anti-oxidants are independently related to CCA-IMT. METHODS AND RESULTS: We investigated cross-sectional relations between CCA-IMT measured by B-mode ultrasound and COX-mediated inflammation (as measured by 15-keto-dihydro-prostaglandin F(2alpha) [PGF(2alpha)], cytokine-mediated inflammation (interleukin-6 [IL-6], high sensitivity C-reactive protein [hsCRP] and serum amyloid A protein [SAA]), oxidative stress (8-iso-PGF(2alpha), an F(2)-isoprostane; a non-enzymatic, free radical-induced product of arachidonic acid), and tocopherols (anti-oxidants) in a small subset of a population-based sample of elderly men (n=234) stating no use of anti-inflammatory medications. In a backward-stepwise regression analysis of correlates of CCA-IMT (with PGF(2alpha), hsCRP, IL-6, SAA, F(2)-isoprostanes, tocopherols, diabetes, body mass index (BMI), beta-blocker, statin treatment, smoking, hypertension and cholesterol), PGF(2alpha), CRP, beta-blocker treatment, diabetes and BMI were independently associated with CCA-IMT. There were no associations between F(2)-isoprostanes or tocopherols and CCA-IMT in this study. CONCLUSION: This study suggests both COX- and cytokine-mediated inflammation to be independently associated with increased CCA-IMT, implying that there might be more than one mode of inflammation involved in atherogenesis.     

Metabolism and elimination of the endogenous DNA adduct, 3-(2-deoxy-beta-D-erythropentofuranosyl)-pyrimido[1,2-alpha]purine-10(3H)-one, in the rat

Endogenously occurring damage to DNA is a contributing factor to the onset of several genetic diseases, including cancer. Monitoring urinary levels of DNA adducts is one approach to assess genomic exposure to endogenous damage. However, metabolism and alternative routes of elimination have not been considered as factors that may limit the detection of DNA adducts in urine. We recently demonstrated that the peroxidation-derived deoxyguanosine adduct, 3-(2-deoxy-beta-D-erythropentofuranosyl)-pyrimido[1,2-alpha]purine-10(3H)-one (M1dG), is subject to enzymatic oxidation in vivo resulting in the formation of a major metabolite, 6-oxo-M1dG. Based on the administration of [14C]M1dG (22 microCi/kg) to Sprague-Dawley rats (n=4), we now report that 6-oxo-M1dG is the principal metabolite of M1dG in vivo representing 45% of the total administered dose. When [14C]6-oxo-M1dG was administered to Sprague-Dawley rats, 6-oxo-M1dG was recovered unchanged (>97% stability). These studies also revealed that M1dG and 6-oxo-M1dG are subject to biliary elimination. Additionally, both M1dG and 6-oxo-M1dG exhibited a long residence time following administration (>48 h), and the major species observed in urine at late collections was 6-oxo-M1dG.     

Levuglandinyl adducts of proteins are formed via a prostaglandin H2 synthase-dependent pathway after platelet activation

The product of oxygenation of arachidonic acid by the prostaglandin H synthases (PGHS), prostaglandin H(2) (PGH(2)), undergoes rearrangement to the highly reactive gamma-ketoaldehydes, levuglandin (LG) E(2), and LGD(2). We have demonstrated previously that LGE(2) reacts with the epsilon-amine of lysine to form both the levuglandinyl-lysine Schiff base and the pyrrole-derived levuglandinyl-lysine lactam adducts. We also have reported that these levuglandinyl-lysine adducts are formed on purified PGHSs following the oxygenation of arachidonic acid. We now present evidence that the levuglandinyl-lysine lactam adduct is formed in human platelets upon activation with exogenous arachidonic acid or thrombin. After proteolytic digestion of the platelet proteins, and isolation of the adducted amino acid residues, this adduct was identified by liquid chromatography-tandem mass spectrometry. We also demonstrate that formation of these adducts is inhibited by indomethacin, a PGHS inhibitor, and is enhanced by an inhibitor of thromboxane synthase. These data establish that levuglandinyl-lysine adducts are formed via a PGHS-dependent pathway in whole cells, even in the presence of an enzyme that metabolizes PGH(2). They also demonstrate that a physiological stimulus is sufficient to lead to the lipid modification of proteins through the levuglandin pathway in human platelets.     

Urinary prostaglandin F2alpha is generated from the isoprostane pathway and not the cyclooxygenase in humans

Prostaglandins (PGs) derived from the enzymatic oxidation of arachidonic acid by the cyclooxygenases (COXs) are potent lipid mediators involved in human physiology and pathophysiology. Structurally similar compounds, the isoprostanes (IsoPs), are generated from the free radical-catalyzed oxidation of arachidonic acid independent of COX. IsoPs exhibit significant bioactivity and play a role in the pathogenesis of diseases associated with oxidant injury. As one of the major PGs, prostaglandin F(2alpha) (PGF(2alpha)) is present in human urine in significant concentrations and is presumed to be derived from COX activity. We determined, however, that levels of putative PGF(2alpha) in urine cannot be suppressed by nonsteroidal anti-inflammatory agents, suggesting that it is generated via another mechanism(s). An important difference between COX-derived PGF(2alpha) and the IsoPs is that the former is an optically pure compound, whereas IsoPs are racemic. Utilizing a rodent model of oxidative stress, we now show that significant amounts of compounds identical in all respects to PGF(2alpha) and its enantiomer, ent-PGF(2alpha), are formed in equal amounts esterified in tissue phospholipids, suggesting that these compounds are derived via the IsoP pathway. Further, employing liquid chromatography/mass spectrometry, the vast majority of putative PGF(2alpha) in human urine is derived from the free radical-initiated peroxidation of arachidonate independent of COX and is composed of PGF(2alpha) and its enantiomer, although the latter compound is approximately 2-fold more abundant. Thus, quantification of urinary PGF(2alpha) actually reflects oxidative stress status as opposed to COX activity. Indeed, levels of this compound are elevated in urine from cigarette smokers and in humans with hypercholesterolemia, two conditions associated with oxidant stress. The elucidation that urinary PGF(2alpha) in humans is derived from the IsoP pathway has implications regarding PG formation and inhibition in vivo.     

Heterocyclic amine-DNA adducts analyzed by 32P-postlabeling method

DNA adducts formed by 12 heterocyclic amines were analyzed by 32P-postlabeling method. Several DNA adducts were detected in rat liver by administration of each heterocyclic amine. Total adduct levels ranged from 0.5 for 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to more than 250 for 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) per 10(7) nucleotides 24 hr after intragastric administration of these compounds. The N-hydroxy derivative of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was reactive toward DNA in vitro to form adducts. Addition of acetic anhydride to N-OH-MeIQx greatly enhanced its reactivity to DNA. 32P-Postlabeling analysis revealed that the MeIQx-DNA adducts formed in vivo and in vitro were identical. Thus, MeIQx would be metabolized in vivo to N-hydroxy form and further esterified to produce more reactive species, such as N-acetoxy form, which modify DNA to form adducts.     

Covalent binding of isoketals to ethanolamine phospholipids

Free radicals have been strongly implicated in the pathogenesis of many human diseases. We previously identified the formation of highly reactive gamma-ketoaldehydes, isoketals, in vivo as products of free radical-induced peroxidation of arachidonic acid. Isoketals react with lysine residues on proteins at a rate that far exceeds that of 4-hydroxynonenal and demonstrate a unique proclivity to crosslink proteins. Hydroxynonenal has been shown to react with aminophospholipids, particularly phosphatidylethanolamine. We explored whether isoketals also react with phosphatidylethanolamine. Using liquid chromatography/electrospray mass spectrometry, we found that isoketals form pyrrole and Schiff base adducts with phosphatidylethanolamine. In addition, the ability of isoketals to covalently modify phosphatidylethanolamine is greater than that of 4-hydroxynonenal. These studies identify in vitro novel isoketal adducts. This provides the basis to explore the formation of isoketal-aminophospholipid adducts in vivo and the biological consequences of the formation of these adducts.     

Histochemical visualization of oxidant stress

Free radicals induce oxidative modification in distinct components of the living matter (lipid, proteins, and DNA). For qualitative and quantitative determination of free radical-induced modifications, different, more or less sensitive biochemical methods are available. Because of the high reactivity and short life of free radicals, ongoing oxidative damage is generally analyzed by measurement of secondary products-such as H(2)O(2), oxidized proteins, peroxidized lipids, and their breakdown products, oxidized DNA-or by fluorographic analysis in combination with fluorescent dyes such as dichlorofluorescin (DCFH). In addition, the determination of free radical-related oxidation products is usually carried out in plasma, urine, or, less frequently, in bioptic material. Consequently, biochemical data seldom reflect the effects of free radical insults in situ. The histochemical visualization of selected molecular markers of oxidative damage can often provide more valuable information concerning the in vivo distribution of oxidative processes. This review summarizes the methods currently available for histochemical detection and indirect visualization of free radical-induced alterations in tissues and isolated cells.